1. Field of the Invention
The field of this invention relates generally to the detection, determination, and quantitation of cyanide compounds, and more specifically, to a cyanide detection method using highly fluorescent and cyanide sensitive boronic acid containing fluorophores, wherein a change in a measured fluorescent property correlates to the concentration of the cyanide compound.
2. Background of the Related Art
Cyanide is one of the most lethal poisons known and the toxicity of its salts has been exploited for many hundreds of years. It was not until 1782 that cyanide was identified, isolated by the Swedish Chemist Scheele, who later died from cyanide poisoning [1]. Blood cyanide levels for healthy persons have been reported as being ≈0.3 μM using a gas chromatography method [4], with lethal cyanide blood levels for fire victims in the cyanide concentration range 23-26 μM [4, 5], some 2 orders of magnitude higher than normal healthy blood levels.
More recently, cyanide was unsuccessfully used as a chemical warfare agent in World War 1, primarily because of the way it was delivered [1]. It is also thought to have been used against the inhabitants of the Kurdish city of Hama, Iraq [2], and in Shahabad, Iran, during the Iran-Iraq war [3]. Based on recent cyanide history, acute cyanide poisoning continues to constitute a threat for military forces in future conventional and unconventional conflicts [1].
Cyanide is also readily used in industry in the making of plastics, in the recovery of gold and silver from ores, and in the electroplating of metals, such as silver, gold, platinum and copper [1]. However, while cyanide is used in both military and industrial applications, cyanide poisoning is not common. However, more surprisingly poisoning occurs from smoke inhalation from residential and industrial fires [1, 4, 5], where the combustion of synthetic products that contain carbon and nitrogen, such as plastics and synthetic fibers, release cyanide. There have been numerous studies of fire victims to assess the lethal levels of cyanide [1, 4, 5, 9]. Fire survivors have been found to have <20 uM cyanide in blood, while victims were found to have levels greater than ≈20-30 uM and in some cases as much as 100 uM cyanide [1, 9].
Cigarette smoke also contains cyanide, the nonsmoker typically averages about 0.06 ug/mL (2.31 uM) of cyanide in blood, where as a smoker typically averages 0.17 ug/mL (6.5 uM) [6].
The mechanism of cyanide poisoning is by absorption. Absorption occurs through the lungs, GI track, and even skin. Cyanide's toxicity lies in its ability to inhibit oxygen utilization by cells, binding the ferric iron in cytochrome oxidase [7, 8], thereby blocking the oxidative process of cells. Hence the tissues with the highest oxygen requirement (brain, heart and lungs) are the most affected by acute poisoning.
The estimated intravenous dose that is lethal to 50% of the exposed population (LD50) of HCN for a man is 1.0 mg/kg, and the estimated LD50 for liquid on the skin is about 100 mg/kg [1]. Hence any cyanide monitoring analytical technique would need a cyanide dynamic range from only few uM to <30 uM to ensure physiological safeguard.
Numerous chemical and physiochemical methods for the detection and determination of cyanides have been used, such as potentiometric, chromatographic, spectrophotometric, flow injection and electrochemical analysis, but only potentiometric determination has been reported as offering continuous cyanide monitoring [48]. Blood cyanide levels for healthy persons have been reported as being ≈0.3 uM using a gas chromatography method [4], with lethal cyanide blood levels for fire victims in the cyanide concentration range 23-26 uM, approximately 100 times higher than normal blood levels. Thus, there are methods for detecting cyanide levels [10-30], but most of these systems are not cheap, portable or field deployable, and most requiring the benefits of an analytical laboratory [10-30].
Fluorescence techniques for sensing a target a fluorescent property, such as lifetime, intensity and wavelength ratiometric sensing [31-33] offer many advantages in the development of miniaturized, cheap, remote, accurate and precise sensors for both laboratory and environmental sensing [31-33]. It is widely accepted that ratiometric or lifetime-based methods offer intrinsic advantages for both chemical and biomedical fluorescence sensing [31, 32]. Fluorescence intensity measurements are typically unreliable away from the laboratory and can require frequent calibration due to a variety of chemical, optical, or other instrumental-related factors. Unfortunately, while fluorescent probes are known to be useful for many applications such as in fluorescence microscopy, fluorescence sensing, and DNA technology, most sensing fluorophores display only changes in intensity in response to analytes and hence relatively few wavelength ratiometric probes are available today.
Some useful wavelength ratiometric probes are available for pH, Ca2+, and Mg2+, but most generally display small spectral shifts and negligible lifetime changes and are subsequently inadequate for quantitive sensing measurements. Thus, one constraint with fluorescence based cyanide sensing to date, has been the development of suitable probes that show appropriate changes in their fluorescent properties in the 100 nM-30 uM cyanide concentration range.
Accordingly, it would be advantageous to develop new methods for determination of cyanide containing compounds that are sufficiently sensitive to quantitatively determine cyanide levels in biological and environmental samples, wherein the method is simple, cheap and fast to both detect and determine cyanide levels up to physiological lethal/safeguard levels, <20 μM, without the negative aspects of the prior art.